Trans10 chemically competent cells

About 2.0 µg RNA from floral buds was treated with a RNase-free DNase I Kit (Promega, USA) in a 10 µl volume to remove genomic DNA contamination. The first-strand cDNA was synthesized with the oligo (dT)₁₈ primers using M-MLV cDNA synthesis kit (Invitrogen, China) following product instructions in a 20 µl volume. Full-length cDNA sequences of the involved genes were obtained by a routine RT-PCR method using gene-specific primers (Supplementary Table S1). Each amplified fragment was purified using the High Pure PCR Product Purification Kit (Roche, Switzerland). Purified fragments were ligated into the pEASY®-Blunt Cloning vector (TransGen Biotech, China) and transformed into Trans10 chemically competent cells (TransGen Biotech, China). Sequencing was performed by Taihe Biotech, China.

We manually searched for target sequences consisting of (N)₂₀GG near the desired mutation sites. The targeted sequences are listed in Table S2. The sgRNAs used for multiple-sgRNA experiments were constructed as previously described (Chen et al. 2014 (link)). The modified sgRNAs ^(F+E), with an extended Cas9 binding structure and the removal of a potential Pol III terminator by an A-U base pair flip, were used in cs-CRISPR experiments (Zhao et al. 2016 (link)). We replaced the unc-119 targeting sequence in the pU6::unc-119 sgRNA^(F+E) expression vector with the desired target sequence using overlap extension PCR (Zhao et al. 2016 (link)). The pU6::unc-119 sgRNA^(F+E) vector was diluted to 2 ng/µl and PCR-amplified to generate linear products. The PCR products were digested by the DpnI restriction enzyme and transformed into Trans10 Chemically Competent Cells (Transgene Biotech, Beijing). We used the PhantaTM Super-Fidelity DNA polymerase (Vazyme Biotech, Nanjing, China, Cat. No. P501-d1/d2/d3) in all PCRs. The primer sequences used for the construction of the sgRNA expression plasmids are listed in Table S3.